A plethora of publications in international journals suggests that cigarette smoke is separated into two phases: a) a solid phase (tar); and b) a gas phase. This separation occurs with the use of a typical Cambridge-glass-fiber filter which withholds 99.9% of the particles which are greater in size than 0.1 .mu.m. The tar of the cigarette contains dramatically high concentrations of very stable free radicals which can be classified into at least four different categories. Semiquinones in equilibrium with quinone and hydroxyquinones are considered to be free radicals with most interesting chemical properties. The quinone system reduces the molecular oxygen to form superoxide (0.sub.2.sup.-) which then upon spontaneous dismutation forms hydrogen peroxide (H.sub.2 O.sub.2). In the gas phase, there are more than 10.sup.15 organic radicals per puff with half-lives of less than 1 second that are inhaled. It is paradoxical however that despite their minute half life these radicals can maintain high levels of activity for more than 10 minutes in the gas phase. In fact the concentration of these radicals is considerably increased as we approach the filter-end of the cigarette. An explanation for this paradox is to be found in the maintenance of a steady state situation; due to the ongoing production of free radicals (Pryor, W. A., Stone, K., Ann. N.Y. Acad. Sci. 686: 12-28, 1993).
Nitric oxide (NO) is the most important free radical in the gas phase of the cigarette smoke which, during smoking, participates in a sequence of reactions through which nitrogen dioxide, isoprene radicals, peroxyl radicals and alkoxyl radicals are formed. Cigarette smoke also contains a considerable number of aldehydes which contribute to its damaging toxic effects. It has been shown that minute amounts of aldehydes extracted from the cigarette smoke cause both protein catabolism and oxidation of thiol groups of the plasma proteins. These properties attributed to the aldehydes are the result of the reactions between the carbonyl group of the aldehydes and the --SH and --NH2 groups of the plasma proteins. For example, acroleine, from the cigarette smoke, reacts quickly with the --SH groups to form carbonyl compounds (Alving, K., Forhem, C., and Lundberg, J. M., Br. J. Pharmacol. 110: 739-746, 1993). In the tar of the cigarette smoke there are trace elements of, for example, iron, copper, manganese and cadmium which are implicated in many free radical producing reactions and lead to the formation of very active secondary radicals (e.g. peroxy radicals, alkoxy radicals, superoxide, cytotoxic aldehydes etc.). The introduction of the trace elements into the lung during cigarette smoking leads to a series of redox reactions both in lung fluids and alveolar macrophages which result in the formation of the very active hydroxyl radicals (OH--). These hydroxyl radicals are mainly formed in the presence of iron via the Fenton reaction. Copper can also form hydroxyl radicals by reacting with the hydrogen peroxide in the lung. Manganese, in low concentrations (10.sup.-7 M), stimulates the soluble guanylate cyclase of the endothelial cells of the lung causing the production of nitric oxide and superoxide through a positive feedback mechanism (Youn, Y. K., Lalonde, C., and Demling, R., Free Rad. Biol. Med. 12: 409-415, 1992). Carbon monoxide is produced during tobacco burning. A quantity of CO is retained in the lung even after exhaling, resulting in the stimulation of the soluble guanylate cyclase after its interaction with the heme moiety of the enzymes of the endothelial cells and other cells of the lung tissue. The increased levels of cyclic GMP within the cells coupled with a positive feed back mechanism increase the production of nitric oxide and superoxide Watson, A., Joyce, H., Hopper, L., and Pride, N. B., Thorax 48: 119-124, 1993). NO gas which can be produced by numerous cell types, including the vascular endothelial cells and reticular endothelial cells, causes relaxation of the smooth muscle (Lowenstein, C. J., Dinerman, J. L., Snyder, S. H. Ann. Intern. Med. 120: 227-237, 1994). There are also exogenous sources of NO which are considered similarly responsible in causing damage to the blood vessels and other tissues. It is well established that secondary and tertiary amines can react with nitrite and other nitrosating agents to form N-nitrosoamines (Lowenstein, C. J., Dinerman, J. L., Snynder, S. H. Ann Intern. Med. 120: 227-237, 1994). Since 1974 a number of studies have demonstrated that during harvesting, tobacco processing and smoking the alkaloids are nitrosated to tobacco specific N-nitrosamines (TSNA). Of the TSNA identified in tobacco and/or its smoke, N-nitrosonornicotine (NNN), 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnirosamino)-1-(3-pyridyl)-1-butanol (NNAL) are strong animal carcinogens. NNN induces tumor of the lung in mice, tumors of the trachea in hamsters, and tumors of the nasal cavity and esophagus in rats. NNK induces tumors of the lung in mice, hamsters and rats, and also tumors of the liver, nasal cavity and pancreas in rats. Oral swabbing of a mixture of NNN and NNK elicits tumors in the oral cavity and lung of rats. The typical amount of both NNK and NNN in mainstream cigarette is 200 ng/cigarette. (Hecht, S. S., Spratt, T. E., and Trushin, N. Carcinogenesis, 9: 161-165, 1988).
Our present research, related to the effect of the cigarette smoke on lung tissue has revealed that NO reacts with superoxide to form the strong oxidant radical peroxynitrite (ONOO--) which causes secondary damaging reactions in key biomolecules. Both the metabolic and damaging effects of the NO in the cells were studied in our lab in vitro and in vivo experiments.
NO is oxidized, in the presence of oxygen, to nitrogen dioxide (NO.sub.2). The rate of this oxidation depends upon the concentration of oxygen and the square of the NO concentration. Nitrogen dioxide is clearly cytotoxic and is transformed into nitrite and nitrate when in water solutions. Moreover NO forms complexes with trace elements and/or with metalloproteins, hemoglobin for example (Wink, D. A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., and Ford, P. E., Chem. Res. Toxicol. 6: 23-27, 1993).
NO that reacts with superoxide to form the noxious compound ONOO.sup.- can justify certain types of superoxide toxicity. ONOO.sup.- is unusually stable, taking into consideration its strong oxidative potential (+1.4 V). During its decomposition it forms strong oxidative derivatives including the hydroxyl radical, the nitrogen dioxide and the nitronium ion. Consequently any modification in the NO and superoxide production by the tissues can lead to the formation of strong secondary oxidative radicals (Deliconstantinos, G., Villiotou, V., Stavrides, J. C., Cancer Mol. Biol. 1: 77-86, 1994). Finally ONOO.sup.- and its esters (RO--ONO or RO--ON0.sub.2) tend to cause inactivation of the alpha -1-proteinase inhibitor (a1PI). This can be justified by the facts that: a) the hydrogen peroxide alone does not cause quick inactivation of the a1 PI but acts only in the presence of NO whereupon ONOO.sup.- is formed and quick inactivation of the a1PI occur, b) solutions of tert-boutyl peroxynitrite (RO--O--O--NO.sub.2) or ONOO.sup.- cause inactivation of a1PI by themselves, and c) amines and amino acids protect the a1PI proteinase from quick inactivation (Moreno, J. J., and Pryor, W. A., Chem. Res. Toxicol. 5: 425-431, 1992). Apart from the free radicals contained in the cigarette smoke the activated alveolar macrophages represent another important source of free radical production by smokers. The alveolar macrophages activated by cigarette smoke undergo a respiration burst resulting in increased production of oxygen free radicals (mainly O.sub.2.sup.-, NO and H.sub.2 O.sub.2). Smokers appear to have an increased number of both alveolar macrophages and circulating neutrophiles. The oxygen free radicals of the cigarette smoke have also been implicated in the development of lung cancer. The inhaled cigarette smoke causes increased oxidative stress in the lung cells resulting in the reduction in the concentration of the intracellular antioxidants. H.sub.2 O.sub.2 reacts, through the production of hydroxyl radicals, with the DNA of the cells and causes a break in the double strand. As this break can be prevented by the addition of catalase, this indirectly confirms the damaging effects of H.sub.2 O.sub.2 and the hydroyl radicals on cellular DNA (Leanderson, P., Ann. N.Y. Acad. Sol. 686: 249-261, 1993). Furthermore H.sub.2 O.sub.2 can cause transformation in the tracheal epithelium of the lung and has been linked to the development of bronchogenic carcinoma in smokers. Thus the detrimental role of H.sub.2 O.sub.2 (contained in the cigarette smoke) in the lung cells and in the development of lung cancer is strongly suggested. The tar from cigarette smoke contains both semiquinone radicals and iron thus creating a system for hydroxyl radical production. The various trace elements contained in the tar of the cigarette smoke (Fe, Cu, Mn, Cd) can act both intracellularly and extracellularly. The Fe.sup.2+ with the well Known Fenton reaction: EQU Fe.sup.2+ +H.sub.2 O.sub.2 .fwdarw.Fe.sup.3+ +OH+OH.sup.-
causes a plethora of oxidative reactions through hydroxyl radicals. Similar production of hydroxyl radical can be achieved by Cd.sup.2+. Mn.sup.2+ is a characteristic stimulator of soluble guanylate cyclase activity. Cd.sup.2+ contained in the cigarette smoke is exceptionally toxic to the lung. Smokers appear to have twice the normal concentration of Cd.sup.2+ in their lungs. It is suggested that Cd.sup.2+ displaces Zn.sup.2+ in presentation of normalcy in the endothelium of the lung vessels (Kostial, K., In: "Trace Elements in Human and Animal Nutrition" (ed. W. Mertz) Fifth edit. Vol. 2: 319-345, Academic Press, Inc. Orlando, Fla., 1986). Aldehydes, present in the cigarette smoke, react with the --SH and --NH.sub.2 groups of the proteins ultimately to become inert. Crotonaldehyde (.alpha., .beta. unsaturated aldehyde contained in cigarette smoke decreases the concentration of the --SH groups and increases the concentration of the carbonyl proteins (Stadtman, E. R., Science 257: 1220-1224, 1991).
Today filters on cigarettes are strongly recommended. The ulimate aim in adding filters to the cigarette is to achieve the maximum retention of noxious compounds present both in the gas and solid phases of the cigarette smoke. Epidemiological studies in smokers have shown that there was a dose-dependent response regardless of whether the cigarette smoke was administered in the gas phase, the solid phase or the solid phase or the combined phase (Surgeon General of the U.S. Public Health Service. The health consequences of using smokeless tobacco, N.H. Publ. No 86-2874, Bethesda, Md., 1986). It was proven that modification of the cigarette is in itself a practical approach to reducing the noxious compounds contained in cigarette smoke. This was initially achieved using common filters and then by changing the composition of the tobacco through chemical processing. Changes in the manufacturing of the cigarettes were also made with the use of porous paper or paper made of tobacco leaves. In the last 15 years many attempts have been made to make smoking less damaging to ones health by: reducing the quantity of the smoke per cigarette: changing the diameter of the cigarette; and by using perforated filters. Perforated filters allow for the dilution of cigarette smoke with air to up to 50%. Activated charcoal has also been used in combination with perforated filters. This has contributed to drastic reduction in smoke yields of tar and nicotine. Such techniques are being used particularly in the developed countries like Austria, Canada, France, Germany, Sweden, England and the U.S.A. The average yield of tar and nicotine in an American cigarette was reduced from 38 mg and 2.7 mg in 1955 to 13 mg and 1 mg in 1991 respectively. In the European Community this trend towards reduction in the yields of tar and nicotine in cigarette smoke is still being continued. The upper allowable limit for tar as of January 1993 is 15 mg which is to be reduced to 12 mg by the beginning of January 1998. However in other countries the yield of tar in cigarette smoke is at 22 mg (Mitacek, E. J., Brun neman, K. D., Pollednak, A. P., Hoffman, D., and Suttajit, M., Prev. Med. 20: 764-773, 1991). The changes made in the manufacturing of cigarettes led to the specific removal of certain toxic substances from the cigarette smoke; more specifically the cellulose acetate filters were introduced thus allowing for the partial removal of the semivolatile phenols and the volatile N-nitrosamines (Brunnemann, K. D., Hoffman, d., Recent. Adv. Tobacco Res. 17: 71-112, 1989). Carbon monoxide is selectively reduced with the use of perforated filters. The concentration of carcinogenic polynuclear aromatic hydrocarbons (PAH) was selectively reduced with the use of tobacco enriched with nitrite. However the reduction of PAH in tobacco using high concentrations of nitrite led to undesirable increases of carcinogenic N-nitrosamines, it was thus necessary to reduce the PAH by alternate means (Hoffman, D., Hoffman, I., Wynder, E.1., Lung Cancer and the Changing Cigarette in Relevance to Human Cancer of N-Nitroso-compounds, Tobacco Smoke and Mycotoxins. (eds. O'Neil, I. K., Chen, J., and Bartsch, H.) Vol. 105: 449-459, 1991).
From the above mentioned it becomes clear that there is a necessity to manufacture a filter capable of withholding the noxious nitrogen oxides, the free radicals, the hydrogen peroxide, the aldehydes, and the carcinogenic nitrosocompounds which are all responsible for the damaging effects of cigarette smoke on the respiratory and cardiovascular systems. For the identification of the noxious compounds contained in the cigarette smoke we have conducted chemical, biological experiments. The chemical experiments performed are the following:
a) Identification and quantitative determination of NO and NOx using a novel chemical and biological method (this method was developed in our lab). PA1 b) Identification of the free radicals using the lucigenine-dependent chemiluminescence methods. PA1 c) Identification of the aldehydes and quinone through stimulation of the enzymatic system luciferine-luciferase (this method was also developed in our lab). PA1 d) Identification and quantitative determination of the trace elements using the method of the oxidation of luciferine by luciferase in the presence of ATP (this method was developed in our lab). PA1 e) Identification and quantitative determination of H.sub.2 O.sub.2 using the isoluminolmicroperoxidase dependent chemiluminescence method. PA1 f) Identification and quantitative determination of ONOO.sup.- spectrophotometrically and by luminol enhanced chemiluminescence method. PA1 g) Identification of the carcinogenic nitroso compound by luminol enhanced chemiluminescence. PA1 a) Identification of NO by using isolated soluble guanylate cyclase activity as functional parameter. PA1 b) Identification of ONOO.sup.- by using the estimation of the oxidative stress of the human erythrocytes induced by ONCO.sup.-. PA1 c) Identification of CO by using isolated soluble guanylate cyclase activity as functional parameter. PA1 a) Isolation of alveolar macrophages from rat lung. PA1 b) Estimation of the oxidative stress of alveolar macrophages induced by tert-butylhydroperoxide (t-BHP). PA1 c) Determination of NO/NO.sub.2.sup.- /ONOO.sup.- produced by alveolar macrophages. PA1 d) Determination of H.sub.2 O.sub.2 produced by alveolar macrophages. PA1 e) Effect of exogenous H.sub.2 O.sub.2 on NO production by alveolar macrophages. PA1 a) Determination of NO in the exhaled air of non-smokers. PA1 b) Determination of NO in the exhaled air of smokers. PA1 c) Determination of NO in the exhaled cigarette smoke. PA1 d) Determination of ONOO.sup.- in the exhaled cigarette smoke. PA1 e) Determination of free radicals in the exhaled cigarette smoke. PA1 f) Determination of aldehydes in the exhaled cigarette smoke.
The Biological experiments performed are the following:
Furthermore we performed the following in vitro experiments:
Experiments in vivo in human volunteers were performed for the determination of the following compounds:
For the determination of NO, NOx contained a) in cigarette smoke, b) released by alveolar macrophages after challenging with cigarette smoke and c) in the exhaled cigarette smoke of human volunteers we designed and fabricated a chamber from 2.5 cm diameter, solid rods of clear Plexiglas which were hollowed out from one end with a machine-lathe to create an identical conical cavity within each of the Plexiglas rods. They were then further machined and polished at the open ends, to form a mated beveled union, creating a very tight fit between the two conical cavities. A thin square of teflon sheet (polytetrafluorethylene 0.0015 inches in thickness) was sandwiched between the assemblies which were recompressed with the thumb-screws. The two tube-access-parts at either side of the membrane, allows biologically active samples and reactive substances to be injected into, withdrawn from or modified at either side of the membrane during biological reactions (FIG. 1).
A. Determination of NO by Chemiluminescence
The standard NO solution was prepared according to the literature (Deliconstantinos, G., Villiotou, V., Fassitsas, C., (1992) J. Cardiovasc. Pharmacol. 12, S63-S65) and (Deliconstantinos, G., Villictou, V., Stavrides, J. C., (1994) In: "Biology of Nitric Oxide", eds. Feelish, M., Busse, R., Moncanda, S., Portland Press, in press). The reaction solution consisted of Hank's Balanced Salt Solution (HBSS) pH 7.4; H2O2 (500 .mu.M); luminol (30 .mu.M) and the total volume was 500 .mu.l. The vial was vigorously stirred and the emission was recorded in Bedrthold AutoLumat LB953 luminometer.
B. Chemical Determination of NO/NO.sub.2.sup.-
The chemical determination of NO was based on the diazotization of sulfanolamide by NO at acidic pH and subsequent oxidation of scopoletin which can be detected fluorometrically as previously described (Deliconstantinos G., Villiotou, V., Fassitsas, C., J. Cardiovasc. Pharmacol 12: S63-S65, 1992). Alveolar macrophages in HBSS (10.sup.6 cells/ml) were mixed with 100 .mu.l of a reagent consisting of: 20% sulfanilamide in 20% H.sub.3 PO.sub.4 and 25 .mu.M scopoletin. The decay of the fluorescence was monitored at room temperature (22.degree. C.) with an Aminco SPF-500 Fluorescence Spectrophotometer. The fluorescence was monitored continuously in time until the slope of the line could be measured (approx. 8 min). Slope measurements were then converted to nmols of NO using a standard curve constructed with various concentrations of pure NO. Nitrite (NO.sub.2.sup.-) the end product of NO synthesis was measured on the basis of their accumulation in the supernatants of cells cultured by its reaction with Griess reagent.
C. Spectroscopical Determination of Peroxynitrite (ONOO.sup.-)
ONOO.sup.- was synthesized, titrated, and stored as previously described (Deliconstantinos, G., Villiotou, V., Stavrides, J. C., In: "Biology of nitric oxide" (eds. Feelisch, M., Busse, R., and Moncada, S.) Portland Press (in press). Because of the instability of ONOO.sup.- at pH 7.4, UV spectra were recorded immediately after mixing the H.sub.2 O.sub.2 and NO solution. The concentration of ONOO.sup.- was determinated based on an .epsilon.302 nm value of 1670 M.sup.-1 cm.sup.-1. UV spectra were shown after subtraction of the basal UV spectra of H.sub.2 O.sub.2 at corresponding concentrations.
D. Estimation of free radicals
The estimation of free radicals was performed by using the lucigenin/DAMCO (1,4 diazabicyclo-2,2,2!octane)-induced chemiluminescence as previous described (Deliconstantinos, G., Krueger, G. R. F., J. Viral Dis. 1: 22-27, 1993). The reaction mixture consisted of HBSS pH 7.4; lucigenin (30 .mu.M); DAMCO (100 .mu.M). The vial was vigorously stirred and the emission was recorded in a Bedthold AutoLumat LB953 luminometer. Scavengers of oxygen free radicals were used (SOD, mannitol, histidine, methionine).
E. Estimation of trace elements and aldehydes
The assays were based on the luciferase-catalyzed oxidation of D-luciferin in the presence of an ATP-magnesium salt according to the reaction: ##STR1##
The trace elements Cd.sup.2+, Cu.sup.2+, Fe.sup.2+ increase the luciferase activity and the maximum chemiluminescence response is proportionally increased according to the concentrations of the trace elements up to 10 .mu.g. The reactions take place in HBSS pH 7.4 in total volume of 0.5 ml.
For the estimation of the aldehydes the same enzymatic system luciferin/luciferase was used but in the absence of ATP. Aldehydes reacts with the enzymatic system to produce chemiluninescence without the presence of ATP. The reagents used were taken from an ATP assay Kit (Calbiochem-Novabiochem Calif., U.S.A.).
F. Isolation of alveolar macrophages
In brief, rats were killed with an intravenous injection of sodium pentobarbital, the thorax was opened, the lungs were perfused free of blood with Ca.sup.2+ free cold (4.degree. C.) phosphate buffered saline (PBS; pH 7.4), and removed intact from the chest cavity. The homogenate of rat lung was obtained by repeatedly drawing the tissue through a syringe and then passing it through successively finer stainless steel screens ranging from 32, 62 and 68 pores per inch., meshes respectively, and under a constant stream of Finkelstein Balanced Salt Solution (FBSS; pH 7.4). The final suspension of alveolar macrophages were pooled, filtered and centrifuged at 300.times.g for 10 min to pellet the cells. The cell pellet, consisting of more than 98% macrophage, was washed and resuspended in Ringer's solution. Then the procedure was repeated two times. Approximately 10.times.10.sup.8 macrophages were isolated per rat. Viability was assessed by trypan blue exclusion.
F. Identification of nitrosocompounds
Nitrosocompounds were identified by the slow release of nitric oxide (NO) after their treatment with H.sub.2 O.sub.2. The reaction solution consisted of dimethyl nitrosamine and/or diethyl nitrosamine (1 .mu.M); H.sub.2 O.sub.2 (500 .mu.M); luminol (30 .mu.M) in HBSS pH 7.4 total volume 0.5 ml. The vial was vigorously stirred and the emission was recorded in a Bedrthold AutoLumat LB953 luminometer. Mannitol (100 mM); DMSO (100 mM) and cysteine (3.0 mM) were used to identifine the formation of ONOO.sup.-.
G. Isolation of alveolar macrophages
In brief, rats were killed with an intravenous injection of sodium pentobarbital, the thorax was opened, the lungs were perfused free of blood with Ca.sup.2+ free cold (4.degree. C.) phosphate buffered saline (PBS; pH 7.4), and removed intact from the chest cavity. The homogenate of rat lung was obtained by repeatedly drawing the tissue through a syringe and then passing it through successively finer stainless steel screens ranging from 32, 62 and 68 pores per inch., meshes respectively, and under a constant stream of Finkelstein Balanced Salt Solution (FBSS; pH 7.4). The final suspension of alveolar macrophages were pooled, filtered and centrifuged at 300.times.g for 10 min to pellet the cells. The cell pellet, consisting of more than 98% macrophage, was washed and resuspended in Ringers solution. Then the procedure was repeated two times. Approximately 10.times.10.sup.8 macrophages were isolated per rat. Viability was assessed by trypan blue exclusion.
H. Oxidative stress of alveolar macrophages induced by t-butyl -hydroperoxide (t-BHP)
The generation of oxygen free radicals by alveolar macrophages induced by t-BHP (2.5 mM) was determined by using a luminol chemiluminescence method. The chemiluminescence response was recorded in a Bedrthord AutoLumat LB953 luminometer as previous described (Deliconstantinos, G., Krueger, G. R. F., J. Viral Dis. 1, 22-27 1993).
I. Determination of hydrogen peroxide (H.sub.2 O.sub.2)
An isoluminol/microperoxidase cocktail (100 mM sodium borate, 1 mM isoluminol, 0.01 mM microperoxidase in 70% water and 30% methanol at pH 8) was prepared. 50 .mu.l of this regent were mixed with the isolated alveolar macrophages (10.sup.6 cells) in HBSS in a total volume of 0.5 ml. The chemiluminscence response was converted to nmols of H.sub.2 O.sub.2 using a standard curve constructed with various concentrations of pure H.sub.2 O.sub.2. J. Preparation and Purification of soluble Guanylate cyclase (sGC) for CO estimation. sGC from human endothelial cells was purified by GTP- agarose chromatography. Cytosols (10 mg protein) were added to a GTP- agarose column (1.8.times.9 cm) pre equilibrated with 25 mM Tris-HCl buffer pH 7.6 containing 250 mM sucrose and 10 mM MnCl.sub.2. sGC was then eluted from the column with 5 ml equilibration buffer plus 10 mM GTP.
K Determination of Cyclic GMP
Concentrations of cGMP were determined by radioimmunoassay after acetylation of the samples with acetic anydride (Delikonstantinos, G., and Kopeikina, L., Anticancer Res. 9: 753-760, 1989). The reaction mixture contained trnethanolamine/HCl (50 mM); creatine phosphate (5 mM); MgCl.sub.2 (3 mM); isobutylmethylxanthine (1 mM); creatine kinase (0.6 Units); GTP (1 mM); soluble guanylate cyclase (1 .mu.g protein) in a total volume of 150 .mu.l. The reactions were initiated by the addition of GTP and incubated for 10 min at 37.degree. C. The incubation medium was aspirated and cGMP was extracted by the addition of ice-cold HCl (0.1M). After 10 min, the samples were transferred to a new plate dried, and reconstituted in 5 mM sodium acetate (pH 4.75) for cGMP determination. cGMP formed was determined using a cGMP assay kit (Amersham).